Quantitative nucleic sequence analysis plays an increasingly important role in the fields of biological and medical research. For example, quantitative gene analysis has been used to determine the genome quantity of a particular gene, as in the case of the human HER-2 oncogene, which is amplified in approximately 30% of human breast cancers. D. J. Slamon et al., Science 235, 177-182 (1987). More recently, gene and genome quantitation have also been used in determining and monitoring the levels of human immunodeficiency virus (HIV) in a patient throughout the different phases of the HIV infection and disease. M. R. Furtado et al., J. Virol. 69, 2092-2100 (1995). It has been suggested that higher levels of circulating HIV and failure to effectively control virus replication after infection may be associated with a negative disease prognosis; in other words, there may be an association between virus level (HIV replication) and the pathogenesis of the disease. M. Paitak et al., Science 259, 1749-1754 (1993). Accordingly, an accurate determination of HIV nucleic acid levels early in an infection may serve as a useful tool in diagnosing illness, while the ability to correctly monitor the changing levels of viral nucleic acid in one patient throughout the course of an illness may provide clinicians with critical information regarding the effectiveness of treatment and progression of disease. Additionally, the determination of virion-associated HIV RNA levels in plasma represents a marker of viral replication with potential widespread applicability in assessment of the activity of antiretroviral therapy. Id.
Several methods have been described for the quantitative analysis of nucleic acid sequences. The polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR) have permitted the analysis of small starting quantities of nucleic acid (i.e., as little as one cell equivalent). See, e.g., S. Edmands et al. 1994, PCR Methods Applic. 3, 317-19; I. R. Rodriguez et al. 1992, Nucleic Acids Res. 20, 3528. Early reports of quantitative PCR report quantitation of the PCR product, but do not measure the initial target sequence quantity. F. Ferre 1992, PCR Methods Applic. 2, 1-9. In general, these methods involve measuring PCR product at the end of temperature thermal cycling and relating this level to the starting DNA concentration. Unfortunately, the absolute amount of product generated does not always bear a consistent relationship to the amount of target sequence present at the initiation of the reaction, particularly for clinical specimens. Such an "endpoint" analysis reveals the presence or absence of starting nucleic acid, but generally does not provide an accurate measure of the number of DNA targets. Id. Both the kinetics and efficiency of amplification of a target sequence are dependent on the starting abundance of that sequence, and on the sequence match of the primers and target template, and may also be affected by inhibitors present in the specimen. In PCR analysis of RNA samples, variable efficiencies in both reverse transcription and amplification steps are potential sources of variability. For these reasons, comparison of the amount of specimen-derived PCR product to the amount of product from a separately amplified external control standard does not provide a rigorous basis for quantitation.
One specific approach to nucleic acid amplification is to measure PCR product quantity in the log phase of the reaction prior to the plateau. See, e.g., Kellogg et al. 1990, Anal. Biochem. 189, 202-208; S. Pang et al. 1990, Nature 343, 85-89. This method requires that each sample has equal input amounts of nucleic acid and that each sample under analysis amplifies with identical efficiency up to quantitative analysis. A gene sequence (contained in all samples at a relatively constant quantity) can be used for sample amplification efficiency normalization. However, using conventional methods of PCR detection and quantitation, it is extremely laborious to assure that all samples are analyzed during the log phase of the reaction, both for the target gene and the normalization gene.
Another method, quantitative competitive PCR (QC-PCR) has been developed and has also been used widely for PCR quantitation. See, e.g., P. D. Siebert and J. W Larrick 1992, Nature 359, 557-558; M. J. Piatak et al. 1993, BioTechniques 14, 70-81; X. Tan et al. 1994, Biochim. Biophys. Acta 1215, 157-162; L. Raeymaekers 1995, Genome Res. 5, 91-94. QC-PCR relies on the inclusion of a known amount of an internal control competitor in each reaction mixture. The efficiency of each reaction is normalized to the internal competitor. To obtain relative quantitation, the unknown target PCR product is compared with the known competitor PCR product, usually via gel electrophoresis. The relative amount of target-specific and competitor DNA is measured; this ratio is used to calculate the starting number of target templates. Basically, in this kind of analysis, the larger the ratio of target specific product to competitor specific product, the higher the starting DNA concentration. Success of a QC-PCR assay relies on the development of an internal control that amplifies with the same efficiency as the target molecule. However, the design of the competitor and the validation of amplification efficiencies require much effort. In the QC-PCR method of RNA quantitation, a competitive RNA template matched to the target sequence of interest, but different from it by virtue of an introduced internal deletion, is used in a competitive titration of the reverse transcription and PCR steps, providing stringent internal control. See, e.g., A. Rashtchian 1994, PCR Methods. Applic. 4, S83-S91. M. Clementi et al. 1993, PCR Methods Applic. 2, 191-196; M. Becker-Andre 1991, Meth. Molec. Cell. Biol. 2, 189-201; R. K. McCulloch et al. 1995, PCR Methods Applic. 4, 219-226. Increasing amounts of known copy numbers of competitive template are added to replication portions of the test specimen, and quantitation is based on determination of the relative (not absolute) amounts of the differently sized amplified products derived from the wild-type and competitive templates, after electrophoretic separation.
In addition to requiring time-consuming and burdensome downstream processing such as hybridization or gel electrophoresis, these assays are limited in dynamic range (i.e. sensitivity to a range of target nucleic acid concentrations). For example, in competitor assays, the sensitivity to template concentration differences may be compromised when either the target or added competitor DNA is greatly in excess of the other. The dynamic range of the assays that measure the amount of end product can also be limited in that the chosen number of cycles of some reactions may have reached a "plateau" level of product prior to other reactions. See, e.g., L. Raeymaekers, supra. Differences in starting template levels in these reactions may therefore not be well-reflected. Furthermore, small differences in the measured amount of product may result in widely varying estimates of the starting template concentration, leading to inaccuracies due to variable reaction conditions, variations in sampling, or the presence of inhibitors.
In an attempt to reduce the amount of post-amplification analysis required to determine initial nucleic acid quantity, additional methods have been developed to measure nucleic acid amplification in "real time." These methods generally take advantage of fluorescent labels (e.g., fluorescent dyes) that are able to indicate the amount of nucleic acid being amplified, and utilize the relationship between the number of cycles required to achieve a chosen level of fluorescence signal and the concentration of amplifiable targets present at the initiation of the PCR process. For example, European Patent Application No. 94112728 describes a quantitative assay for an amplifiable nucleic acid target sequence which correlates the number of temperature cycles required to reach a certain concentration of target sequence to the amount of target DNA present at the beginning at the PCR process. In this assay system, a set of reaction mixtures are prepared for amplification, with one preparation including an unknown concentration of target sequence and others containing known concentrations (standards) of the sequence. The reaction mixtures also contain a fluorescent dye that fluoresces when bound to double-stranded DNA. The reaction mixtures are thermally cycled in parallel for a number of cycles to achieve a sufficient amplification of the targets. The fluorescence emitted from the reaction mixtures is monitored in real time (i.e. as the amplification reactions occur), and the number of cycles necessary for each reaction mixture to fluoresce to a arbitrary intensity threshold (arbitrary fluorescent value, or AFV) is determined. The number of cycles necessary (CT) for the mixture of unknown nucleic acid to reach the AFV value is then compared to the number of cycles necessary for the known mixtures to reach the AFV value. This method relies on a direct correlation between the number of cycles necessary to achieve a given fluorescence intensity, and the logarithm of concentration of the nucleic acid targets. This relationship is thus used to obtain the initial quantity of target nucleic acid sequence in the mixture of unknown concentration. The method provides for the selection of an arbitrary threshold from which to measure numbers of amplification cycles, in that recorded amplification profiles (amplification curves) are analyzed to find an AFV in which to compare the unknown amplification profiles with the standard amplification profile. However, no particular significance is placed on the initial selection of the AFV or cutoff value, in that an exemplary AFV value is chosen to be in the middle of a range in which all the amplification curves are relatively straight (i.e. in transition from upward exponential amplification to downward curving to an asymptote). Moreover, similar results in nucleic acid quantitation are reported for a fairly broad range of AFV values. One shortcoming of this known method is that below an initial copy number of 10.sup.3 target sequence molecules, the method is unreliable in accurately determining initial concentration. Another shortcoming of this method is that it assumes that the shape of each amplification curve remains the same, regardless of whether or not it really does. Finally, only a few data points near the arbitrary cutoff value are taken to determine the initial target nucleic acid concentration.
C. A. Heid et al. 1996 (Genome Research 6, 986-994) and U. Gibson et al. 1996 (Genome Research 6, 995-1001) report methods of real-time quantitative PCR for DNA and RNA quantitation analysis, respectively. Both assays utilize dual-fluorescence reporter systems and are based on the use of the 5'-nuclease assay described by Holland et al. 1991, Proc. Natl. Acad. Sci. USA 88, 7276-7280. In these methods, a computer algorithm generates an amplification plot by comparing the amount of reporter dye emission with the number of amplification cycles that have occurred. The algorithm calculates the cycle (C.sub.T) at which each PCR amplification reaches an arbitrarily selected (i.e. usually 10 times the standard deviation of the baseline) threshold or cutoff. The relative fluorescent emission threshold is based on the baseline of the reporter dye emission during the first 10-15 amplification cycles. It was demonstrated that the calculated C.sub.T value is proportional to the number of target copies present in the sample. Thus, the C.sub.T value is found to be a quantitative measurement of the copies of the target found in any sample.
As indicated by the foregoing discussion, the prior art teaches that one way to perform quantitative analysis of real-time nucleic acid amplification is to select a number of readings just above and below a chosen cutoff level, fit a linear regression to these points, and solve for the "cycle number" at which the fitted line crosses the cutoff level. The selection of a cutoff level is accordingly somewhat arbitrary, as in selecting a cutoff level about 10 times that of a baseline value, or the maximum value of non-target containing samples, or a certain cutoff level above what has been considered acceptable "noise." One shortcoming of such a cutoff selection criteria is that if the cutoff value corresponds to a time point in the amplification during which the amplification rate is changing (i.e., early in the amplification), a linear fit to the data will be inappropriate.
It has heretofore not been appreciated that small differences in the selection of cutoff levels used in quantitation algorithms may have a substantial effect on the ultimate quality (i.e., accuracy) of quantitation. There remains a need to provide an objective and automatic method of selecting preferred cutoff values that will allow users of amplification methods to determine the initial concentrations of target nucleic acids more accurately and reliably than present methods. There also remains a need to provide for methods of determining dynamic cutoff levels that can be easily varied from one experiment or determination to another.